Non-phosphorylated Tyr-1248 form of human epidermal growth factor receptor 2 (HER2) predicts resistance to trastuzumab therapy and poor disease-free survival of HER2-positive breast cancer patients

Aim To determine the predictive value of phosphorylated human epidermal growth factor receptor 2 (pHER2Y1248) status in breast cancer (BC) patients undergoing trastuzumab-based adjuvant therapy. Methods Immunohistochemical status of pHER2Y1248, EGFR/HER1, HER3, and HER4 was determined in 124 consecutive HER2-positive BC patients (median age [range] = 57 years [49.0-64.0]) treated at the University Hospital for Tumors, Zagreb, between 2008 and 2011. The median follow-up was 84 months (60.0-84.0). Prognostic factors of disease free survival (DFS) rate were evaluated with Kaplan-Meier/log-rank test and Cox regression analysis. Results pHER2Y1248, HER1, HER3, and HER4 were expressed in 66.1%, 9.7%, 70.2%, and 71.0% of patients, respectively. Disease progression (DP) was observed in 17.1% of pHER2Y1248-positive and 47.6% of pHER2Y1248-negative BCs (P = 0.001). Kaplan-Meier analysis showed a worse five-year DFS in pHER2Y1248-negative patients who were older than 60 years (P < 0.001) and had positive lymph node status (P < 0.001); tumor size >2.0 cm (P < 0.001); higher histological grade (P < 0.001); HER2E intrinsic subtype (P < 0.001), negative hormone receptors (P < 0.001); negative HER1 status (P < 0.001), positive HER3 (P = 0.002); and/or positive HER4 (P = 0.002) status. The only negative prognostic factor for five-year DFS in multivariate Cox regression analysis was pHER2Y1248-negative (hazard ratio [HR] 3.6, 95% confidence interval [CI] 1.8-7.2, P < 0.001) and lymph node-positive status (HR 3.6, 95% CI 1.3-9.8, P = 0.014). Conclusion pHER2Y1248 predicts sensitivity to trastuzumab and a better five-year DFS regardless of any other prognostic parameter. In HER2-positive BC patients. Non-phosphorylated HER2Y1248 is a strong predictor of trastuzumab resistance and a poor DFS.

Until the development of trastuzumab, a highly-specific monoclonal antibody targeted against human epidermal growth factor receptor 2 (HER2), breast cancer (BC) with positive HER2 was an aggressive and rapidly proliferating malignancy with a poor prognosis. HER2 is overexpressed in about 20% of BC patients, and trastuzumab reduces the risk of disease recurrence almost by half (1)(2)(3)(4). However, a subset of HER2-positive BC patients fails to benefit from such therapy (1)(2)(3)(4)(5)(6). Resistance to trastuzumab-based therapy was recorded in almost 30% of patients, and different resistance mechanisms have been described (1)(2)(3)(4)(5)(6)(7). Besides, HER2-positive BC is very heterogeneous and includes tumors with positive (luminal type) and those with negative (HER2-enriched type) estrogen-(ER) and progesterone-(PgR) hormone receptor status (5). Thus, the approach to BC treatment is not uniform, and the equal response to therapy or the same mechanisms of resistance cannot be expected (4)(5)(6)(7). HER2 is a member of the epidermal growth factor receptor (ErbB) family of four human receptor tyrosine kinases (ErbB1/EGFR/HER1, ErbB2/HER2, ErbB3/ HER3, and ErbB4/HER4). Ligand binding on the extracellular receptor domain leads to its homo-or hetero-dimerization, resulting in its activation/phosphorylation on the receptors' cytoplasmic domain (4,6,8,9). Although without a known ligand, HER2 is the preferred and the most potent dimerization partner due to its high catalytic activity. Active HER2 has several phosphorylation sites. In cases of overexpression (due to gene amplification as in HER2-positive BC), tyrosine-1248 (pHER2 Y1248 ) is the most potent site because it is constitutively activated as a consequence of HER2 homodimerization. Other phosphorylation sites are usually activated by heterodimerization (6,8,9). So far, HER2 status is the only validated biomarker for anti-HER2 therapy in BC patients (1)(2)(3). However, detection of gene amplification or protein overexpression may not truly reflect the activated status of HER2. We assumed that the phosphorylation status of HER2 was a true indicator of its activity and that its heterodimerization with other ErbB family members might contribute to trastuzumab resistance. Therefore, the study aimed to evaluate the predictive value of pHER2 Y1248 coexpressed with other ErbB family members and hormone receptors in HER2-overexpressing BC patients postoperatively treated with trastuzumab-based therapy.

Patients
This retrospective study was performed on treatment-naive, archived formalin-fixed paraffin-embedded (FFPE) tu-mor tissues surgically removed from 124 consecutive patients diagnosed with HER2-positive primary ductal invasive breast cancer (BC). The patients were treated at the University Hospital for Tumors, Zagreb, between 2008 and 2011. All patients received adjuvant trastuzumab-based therapy for at least one year. Demographic and clinicopathological data were retrieved from medical records. Disease-free survival (DFS) rate was defined as the time in months from the date of surgery to the date of disease progression (DP). Data on disease progression, revealed by radiological methods (ultrasound, magnetic resonance, or positron emission tomography-computed tomography) as local recurrence or distant metastases, were obtained from the clinical database. Patients with other complications during trastuzumab therapy were not included. The follow-up period was over 84 months with the last check-up performed in May 2018. All participants signed the informed consent. The study was approved by the Ethics Committee of the University Hospital for Tumors (EP-15506/11-6).
Immunohistochemical staining pHER2 Y1248 and ErbB family members were immunohistochemicaly analyzed on FFPE samples prepared as tissue microarray blocks (Tissue-Tek®Quick Ray System; Sakura, Japan). All BC samples are routinely processed immediately after surgery to avoid the potential loss of epitopes due to a delayed time to fixation. FFPE blocks were processed as previously described (10). Shortly, 3-μm thick serial microarray tissue sections were heated in a water bath for 20 min at 97 °C in an antigen retrieval solution, pH 9.0 (S2367; Dako, Glostrup, Denmark). The sections were incubated (overnight, 4 °C) with primary antibodies (pHER2 Y1248  The expression of pHER2 Y1248 was assessed according to the semiquantitative HercepTest scoring method (11,12). BCs expressing moderate to strong membranous staining in more than 10% of cells were considered pHER2 Y1248 -positive (10). Tumors without staining, with weak membranous staining, or with only cytoplasmic staining were considered pHER2 Y1248 -negative.
The HercepTest method was also applied for immunohistochemical analysis of EGFR/HER1, HER3, and HER4 with BCs exhibiting 2+ or 3+ membranous/cytoplasmic staining considered as positive.

Statistical analysis
The normality of distribution was assessed with the Shapiro-Wilk test. Data are presented as frequencies or median and interquartile range (IQR). Relationships between immunohistochemical data and clinicopathological parameters were assessed with the Spearman correlation (r), t test, and χ 2 , or Fisher exact test. The Kaplan-Meier/log-rank test was used to assess the difference in five-year DFS rate between the patient subgroups. Univariate and multivariate Cox proportional hazards model was used to determine the independent prognostic effect of individual variables on DFS rate, with the results presented as hazard ratio (HR) and 95% confidence interval (CI). All statistical tests were two-sided. The intergroup differences with α<0.05 were considered significant and corrected according to the Bonferroni procedure (the corrected level of significance is Pc = 0.05/N; N-number of independent tests). Statistical analysis was performed with SPSS, trial version (IBM Corp., Armonk, NY, USA).

Patients' characteristics
The median age at the time of surgery was 57 years. The majority of patients were younger than 60 years (62.1%). Tumors >2.0 cm were present in 59.7% of patients. BC samples were mostly classified as histological grade III (56.45%) and were associated with positive axillary lymph nodes (60.5%) at the time of surgery ( Table 1).
The median follow-up was 84 months, and 34/124 (27.4%) patients had disease progression (DP). Among them, the median time to DP was 23.5 months (IQR 18.0-34.5). Positive expression of ER and PgR was detected in 54% and 40.3% of patients, respectively (Table 1). Thus, patients were classified in two intrinsic subtypes: luminal B (ERand/or PgR-positive) and HER2E (ER-and PgR-negative), with 56.5% of patients belonging to the luminal B subtype (Table 1).
Among the standard clinicopathological characteristics, DP positively and weakly correlated with positive lymph node status (r = 0.31; P < 0.001) and higher histological grade (r = 0.25; P = 0.005) ( Table 4). However, after the Bonferroni correction the relationship remained significant only for positive lymph node status ( Table 4).
The average DFS of pHER2 Y1248 -negative patients older than 60 years, with higher histological tumor grade, a larger tumor, or positive lymph nodes was 54.8, 47.2, 46.2, and 44.9 months, respectively ( Figure 5 and 6).
Univariate Cox regression analysis revealed that the intrinsic subtype, hormone receptor status, and HER3 and HER4 staining were not significantly related to DFS ( Table 5). Nevertheless, when these biomarkers were stratified according to pHER2 Y1248 status, Kaplan-Meier analysis showed significant differences in five-year DFS (P < P c [0.05/11 = 0.005]) ( Figure 6). Thus, pHER2 Y1248 -negative patients with HER2E intrinsic subtype, as well as those with HER1-negative staining (Figure 7) or negative hormone receptors ( Figure 8) had a worse five-year     DFS. The same was true for pHER2 Y1248 -negative patients with the coexpression of HER3+ or HER4+ (Figure 9).

dISCUSSIon
In our study, pHER2 Y1248 predicted sensitivity to trastuzumab and a better five-year DFS regardless of any other prognostic parameter. Additionally, pHER2 Y1248 -negative and lymph node-positive status were the only negative prognostic factors for five-year DFS of HER2-positive BC patients. Furthermore, non-phosphorylated HER2 Y1248 was a strong predictor of resistance and poor five-year DFS in combination with any clinicopathological parameter: in patients older than 60 years or those with positive lymph nodes, larger tumor size, or higher histological stage. This was especially true in patients with HER2E-type tumor or those negative for EGFR/HER1 and/or positive for HER3 or HER4.
The expression of pHER2 Y1248 receptors was identified in 66.1% of our patients, consistent with 68% of HER2-positive BC cases in the report by Cicenas et al (13). A relatively high expression rate of pHER2Y 1248 in HER2-positive BC was also found by other researchers (14-19). However, smaller rates (12%-38.2%) were also reported (20)(21)(22)(23)(24)(25)(26)(27). The discrepancies in pHER2 Y1248 expression might be explained by dif-ferences in assays sensitivity, scoring systems, cut-off values used for the evaluation of pHER2 expression, and likely difference in the degree of phosphorylation/activation of HER2 in biologically distinct BC patient cohorts. Thus, for example, HER2 overexpression with concomitant pH-ER2 Y1248 -positive status was considerably less common in ER+ BCs than previously believed (28).
Taniyama et al (19) showed pHER2 Y1248 expression to be highly specific for HER2 gene amplification, advanced tumor stage, and poor DFS of patients with invasive ductal BC. Furthermore, the total level of pHER2 Y1248 is considered to be physiologically more important than the overall number of HER2 present in the cancer tissue (20). Importantly, not all BC patients with pHER2 Y1248 also overexpressed HER2, which indicates signaling activation distinct from the classical pathway involving homo-dimerization and hetero-dimerization with other ErbB family members (13,29). Previous studies also indicate that phosphorylation of HER2 at Y1248 occurs only when HER2 is dimerized and activated irrespective of its overexpression status (11,24).
In our study, patients with positive pHER2 Y1248 had a nearly three times lower risk of DP after trastuzumab-based treatment and a better five-year DFS compared with patients with negative pHER2 Y1248 , of whom 58% relapsed.
Similarly, Hudelist et al (39) reported that pHER2 Y1248 -positive staining was the only covariate predicting the benefit of trastuzumab-based treatment in metastatic BC patients exhibiting moderate or strong HER2 overexpression (39). Notably, in their cohort, the progression-free survival to trastuzumab-based treatment was more than doubled in pHER2 Y1248positive compared with pHER2 Y1248 -negative BC (39).
Better response of pHER2 Y1248 -positive BC to trastuzumabbased therapy was also reported by Giuliani et al (27). Besides, Dokmanovic et al (40) reported that pHER2 Y1248 -positive staining in HER2-positive BC correlated with increased trastuzumab response in the neoadjuvant settings. Notably, the majority of the patients with DP or residual disease after neoadjuvant trastuzumab treatment were pH-ER2 Y1248 -negative, whereas 4/5 patients with complete or near-complete pathological remission were pHER2 Y1248positive (40).
A trend toward increased sensitivity to trastuzumab treatment was also confirmed in experiments on HER2-overexpressing BC cell lines (9). In the study by Ginestier et al (9), the majority of cell lines (8/10)  Interestingly, Cheng et al (46) found no association between pHER2 Y1248 and the response to trastuzumab-containing neoadjuvant therapy in the pre-surgical setting.
In our study, the majority of HER2-positive patients with DP exhibited hormone receptor-negative, EGFR/HER1negative immunostaining, and positive HER3 and/or HER4 status. In a study by DiGiovanna et al (14), pHER2 Y1248 expression was significantly associated with a positive expression of EGFR/HER1. A worse outcome was observed in patients who were either positive for all three biomarkers examined (pHER2 Y1248 , EGFR/HER1, and HER2) or positive only for EGFR/HER1 and HER2 but negative for pH-ER2 Y1248 (14). Significantly higher expression of pHER2 Y1248 among patients with high EGFR/HER1 levels was also reported by Cicenas et al (13). However, despite the positive correlation between the pHER2 Y1248 -positive status and EGFR/HER1, its overall expression did not differ between pHER2 Y1248 -positive and pHER2 Y1248 -negative patients. In addition, pHER2 Y1248 expression positively correlated with HER2 and inversely correlated with hormone receptors, HER3, and HER4. Hormone receptors and HER4 levels were significantly lower in patients with higher pHER2 Y1248 (13). Contrary to these reports, Kurebayashi et al (18) reported no significant correlation of pHER2 Y1248 with EGFR/HER1 and HER4 expression in HER2-positive BC patients. However, pHER2 Y1248 significantly correlated with HER2 and HER3 expression (18).
In our study, a worse five-year DFS was detected in patients with pHER2 Y1248 -negative and either hormone receptor-negative or HER1-negative status and in patients with pHER2 Y1248 -negative immunostaining co-expressed with positive HER3 or HER4. Our results suggest that in such cases phosphorylation occurred at other phosphorylation sites and heterodimerization activated other signaling pathways.
There are several limitations to our study. The sample size was moderate, encompassing BC patients with specific biomarker signatures. Second, the majority of BC in our patient cohort exhibited a HercepTest score of 3+, while the number of HER2 equivocal (2+) patients was limited. Third, all patients received adjuvant trastuzumab therapy so future studies should assess the prognostic value of pH-ER2 Y1248 in the neoadjuvant settings.
In conclusion, our results indicate that the pHER2 Y1248negative status of HER2-overexpressing BC repre-sents a strong independent predictor of tumor resistance to trastuzumab-based therapy and poor five-year DFS rate irrespective of other biomarkers and clinicopathological variables tested. Further studies are needed to investigate the predictive value of the pHER2 Y1248 in a larger cohort of HER2-positive BC patients.
Funding None.
ethical approval given by the Ethics Committee of the University Hospital for Tumors (EP-15506/11-6).
Declaration of authorship SR and FK conceived and designed the study; all authors acquired the data; all authors analyzed and interpreted the data; SR and FP drafted the manuscript; all authors critically revised the manuscript for important intellectual content; all authors gave approval of the version to be submitted; all authors agree to be accountable for all aspects of the work.

Competing interests All authors have completed the Unified Competing
Interest form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare: no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; no other relationships or activities that could appear to have influenced the submitted work.